My research is focused on Preventing dementia by administrating certain compounds in animals. Prior, to that I like to screen my compounds in SK-N-SH cell line by creating a cytotoxic condition using amyloid beta. But I do no how to dissolve, dilute and induce beta amyloid in cell line? There are many manuscripts regarding this topic but I cant get standard and step by step protocol.
Useful!
1. gsbooks.gs.kku.ac.th/52/ngsr12/file/MMP13.pdf
by J Jantaravinid -
β-amyloid peptide is the main content of senile amyloid plaque, which is the key role for the ... Aβ(1-42)- induced oxidative cell death in SK-N-SH neuroblastoma cells. .... solution was diluted to various concentrations ... SK-N-SH human neuroblastoma cell line was ... dissolved in DMSO and measured by microplate reader.
2. Protective Effects of Mangosteen Extract on H2O2-Induced ...
www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
by J Sattayasai - 2013 - Cited by 3 - Related articles
Dec 27, 2013 - Protective effects of ME against β-amyloid peptide (Aβ), induced cytotoxicity have been reported. ... In SK-N-SH cell cultures, 200 μg/ml ME could partially antagonize the .... Germany) was freshly diluted in phosphate buffer saline solution ... Formazan crystals formed by living cells were then dissolved in ...
I used to work with Ab1-42, Ab1-40 and Ab25-35.
You can dissolve all of the in DMSO or in buffer. DMSO amyloid mix should be diluted at least 100x with the buffer. Here is one example for Ab1-42: "Fibrils were generated by dissolving the peptides in PBS buffer (10 mM K-phosphate,
pH 7.4, 140 mM NaCl ) at 0.5 mg/ml concentration. Ab1–42 fibrils were grown in solution at room temperature for several days." (Karsai et al. Journal of Structural Biology 155 (2006) 316–326). You can use sonication to help dissolving the peptides.
You can dissolve Ab25-35 in water and than dilute it in cell culture medium to have the appropriate concentration. They are toxic in the uM range.
See the attached file
The tricky part is that after dissolution the fibrils start growing with various kinetics. Ab1-42 and Ab25-35 is relatively faster (few days to form fibrils) while Ab1-40 is slower (week).
If you want to track the fibril formation to verify what amyloid species are present (oligomers or protofibrils or fibrils) you must use AFM or TEM.
You can get general idea by monitoring the fluorescent signal of Thioflavin-T which has to increase parallel with amyloid formation .
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